Teratogenesis, the formation of congenital defects, was recognized early in this century as an illness and as an important cause of morbidity and mortality among newborns. In 1928 Murphy published a study in which he followed 320 human pregnancies and found 14 cases of children with small head circumference and mental retardation. The defect was ascribed to therapeutic irradiation of the mothers of these children early in their pregnancies. Viruses and nutritional deficiencies were also shown to be potentially teratogenic.
The drug thalidomide, a sedative-hypnotic, was introduced in the late 1950s. Use of this drug by pregnant women resulted in some 10,000 babies suffering from phocomelia, a normally rare congenital malformation with shortening or absence of limbs. The impact of the teratogenic effect of this drug led society to attempt to actively prevent the introduction for human consumption of teratogenic drugs. It became mandatory to subject new drugs, foods, pesticides, or contaminants to different types of tests to determine their potential teratogenicity.
The standard practice for testing a new drug for teratogenicity typically involves studies with more than one kind of animal. Mammalian species such as rats, rabbits, mice, and hamsters are commonly used. Pigs are sometimes used because of their relatively close phylogenetic relationship with humans, and because their diet is similar to that of humans. Dogs, cats, and nonhuman primates have also been employed. These available assays are time-consuming and expensive. In recent years, societal pressure has increased dramatically to limit and, wherever possible, find substitutes for tests involving vertebrate and particularly mammalian animals.
Teratogen assays involving fruit-fly cells has been previously described. The preparation of primary embryonic cell cultures from 3 to 8 hour old Drosophila (fruit-fly) embryos has been described in some detail (Seecof, R. L., Tissue Culture Association Manual 5, 1019-1022, 1979). Cells in these cultures differentiate in vitro to myotubes and ganglia. A teratogen assay based on inhibition of these differentiation processes has been reported (Bournias-Vardiabasis, N., and R. L. Teplitz, Teratog. Carcinog. Mutagen. 2:333-341, 1982; Bournias-Vardiabasis, N., et al., Teratology 28:109-122, 1983). This inhibition would have to be assessed by microscopic observation, and so this assay would be semiquantitative as well as tedious to perform.
Heat shock genes are a group of genes that occur in apparently all living organisms and that are typically silent at the normal temperature of growth of the organism but are activated at somewhat elevated temperatures. (For a review, see: Schlesinger, M. F., et al., ed., Heat shock: From bacteria to man, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982). The genes have been discovered in the fruit fly Drosophila (Ritossa, F., Experientia 18:571-575, 1962). The fly genes are silent at moderate temperatures but become very active at temperatures between 35.degree. and 38.degree.-39.degree. C.
Heat shock genes were cloned initially from Drosophila and more recently also from many other organisms (see Craig, E. A., CRC Crit. Rev. Biochem. 18:239-280, 1985, for a review). Drosophila melanogaster has one gene encoding an 83 kDa heat shock protein (hsp83), 5 genes encoding the major 70 kDa heat shock protein (hsp70), and one gene encoding each of four small heat shock proteins with molecular weights of 27, 26, 23, and 22 kDa (hsp27, hsp26, hsp23, and hsp22). The small heat shock protein genes are also highly active (at normal temperatures) during certain distinct stages of normal development; they are all expressed at relatively high levels in late third instar larvae, during which the titer of the molting hormone ecdysterone is maximal (Sirotkin, K., and N. Davidson, Dev. Biol. 89:196-210, 1982; Mason, P., et al., Mol. Gen. Genet.194:73-78, 1984). This finding suggested that the small heat shock genes may be regulated directly or indirectly by ecdysterone. Strong evidence for this was provided by experiments that demonstrated activation of the genes in cultured Drosophila cells or isolated imaginal discs following the addition of ecdysterone to the medium (ireland, R., et al., Dev. Biol. 93:498-507, 1982).
Buzin and Bournias-Vardiabasis recognized that the same set of drugs that inhibited differentiation of embryonic Drosophila cells in vitro, also caused activation of two of the small heat shock protein genes, encoding the 23 and 22 kDa protein species. In these experiments heat shock protein synthesis was monitored by .sup.35 S-methionine-labeling of proteins and autoradiography of two-dimensional electrophoresis gels (Burzine, C. H., and N. Bournias-Vardiabasis, in Heat shock: from bacteria to man, M. J. Schlesinger, et al., eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 389-394, 1982; Proc. Natl. Acad. Sci. USA 81:4075-4079, 1984).
By the mid-1980s, at least three methods were available for transfection and measuring transient expression of genes in Drosophila cells (Rubin, G. M., and A. C. Spradling, Science 218:348-353, 1982; DiNocera, P. P., and I. P. Dawid, Proc. Natl. Acad. Sci. USA 80:7095-7098, 1983; Lawson, R., et al., Mol. Gen. Genet. 198:116-124, 1984).
The Drosophila melanogaster 23 kDa heat shock protein gene has been cloned and characterized by Voellmy et al. (Cell 23:261-270, 1981). Segments containing extensive 5' nontranscribed sequences from the 23 kDa heat shock protein gene, the RNA leader region, and part of the protein-coding region were linked in frame to an E. coli .beta.-galactosidase gene. The resulting constructs encode a hybrid protein with .beta.-galactosidase activity that is made under the control of the linked heat shock gene sequences (Lawson, R., et al., Dev. Biol. 100:321-330, 1985). These constructs were introduced into Drosophila melanogaster S3 cells (an established cell line) by transfection (Lawson, R., et al., Mol. Gen. Genet. 198:116-124, 1984). The transfected hybrid genes produced E. coli-specific .beta.-galactosidase in response to heat treatment or ecdysterone addition to the medium (Lawson, R., et al., Dev. Biol. 110:321-330, 1985; Mestril, R., et al., EMBO J. 4(11):2971-2976, 1985; Mestril, R., et al., EMBO J. 5(7): 1667-1673, 1986).
The established procedure of P-element-mediated transduction of genes in Drosophila melanogaster has been described in detail (Rubin, G. M., and A. C. Spradling, Science 218:348-353, 1982) has is frequently used to introduce genes of interest into the germline. A hybrid gene that consists of the Drosophila heat shock gene, hsp70, fused to the E. coli .beta.-galactosidase gene has been introduced into the Drosophila germline by the P-element microinjection method. The .beta.-galactosidase activity in the transformants was reportedly inducible by heat shock and showed a widespread distribution throughout the tissues of larvae and adults (Lis, J. T., et al., Cell 35:403-410, 1983).